The genome sequence of a pipunculid fly, Nephrocerus scutellatus (Macquart, 1834)

We present a genome assembly from an individual female Nephrocerus scutellatus (pipunculid fly; Arthropoda; Insecta; Diptera; Pipunculidae). The genome sequence is 613.4 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 18.18 kilobases in length.


Background
Nephrocerus scutellatus is a parasitoid in the family Pipunculidae or Big-headed Flies (Motamedinia et al., 2022).The Pipunculidae are distinguished from other flies by the wing venation and large compound eyes, which give them the appearance of having a large spherical head (Kozánek & Belcari, 1997).Nephrocerus scutellatus is distinguished from other members of the genus by its large size, wing length 8.0-9.5 mm, yellow scutellum, entirely black arista, small brown third segment of the antennae, and hind tibia which is not broadened at the tip and lacks an apical fringe of bristly hairs (Coe, 1966;Johnson, 1915).
Nephrocerus scutellatus is primarily a European species, of central and southern Europe (Coe, 1966) but it is also found in Scandinavia (de Meyer, 1992).It was first recorded in the United Kingdom in 1980(Chandler, 2023;Stubbs, 1980).
The developing larvae of Pipunculids feed upon the bodies of their insect hosts and the females have heavily sclerotised ovipositors that allow them to penetrate their hosts' exoskeletons.Most Pipunculids are host specific but, although there are differences in the structure of the ovipositor between species, these do not correlate with the type of host (Kozánek & Belcari, 1997).While most Pipunculidae are thought to be parasitoids of leafhoppers (Hemiptera: suborder Auchenorrhyncha), the larger flies in the genus Nephrocerus parasitise craneflies (Diptera: Tipulidae) (Koenig & Young, 2007).A single larva of Nephrocerus scutellatus was found developing inside a female Tipula (Lunatipula) helvola (Kehlmaier & Floren, 2010).
We present a chromosomally complete genome sequence for Nephrocerus scutellatus based on one adult female specimen collected in Wytham Woods, Oxfordshire.

Genome sequence report
The genome was sequenced from one female Nephrocerus scutellatus (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.76,.A total of 40-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 89 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 82.00%, and increasing the scaffold N50 by 19.50%. The final assembly has a total length of 613.4 Mb in 8 sequence scaffolds with a scaffold N50 of 119.9 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.98%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 5 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/566305.Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sample acquisition and nucleic acid extraction
RNA was extracted from abdomen tissue of idNepScut1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Markus Friedrich
Wayne State University, Detroit, Michigan, USA This manuscript reports the successful chromosome-level sequencing of the genome of the pipunculid fly species Nephrocerus scutellatus by the Wellcome Sanger Institute.The experimental and computational workflow are described in concise detail.This new high quality genome sequence will be another important contribution to the fields of comparative genomics and biodiversity studies.
Minor comment: "is" in first sentence of the Background section needs to be de-italicized.
Is the rationale for creating the dataset(s) clearly described?

Jerome H L Hui
The Chinese University of Hong Kong, Hong Kong, Hong Kong In this data note, Falk and colleagues sequenced and assembled the genome of a dipteran species, commonly known as big-headed fly, Nephrocerus scutellatus (Macquart, 1834).This species has been reported in southern England according to Falk and Chandler 2005 (Joint Nature Conservation Committee, ISSN 1473-0154).Molecular data of this species are scarce prior to this report, and are mainly COI sequences deposited to the NCBI database.Therefore, this new genome resource will be useful for further studies, ranging from studying the mechanism of being parasitoids to insect hosts, their population structure, to understanding their evolution with other insects.
This genome resource is excellent from the summary statistics, with high BUSCO number scores, high sequence continuity (scaffold N50), and majority of sequences contained on the 5 pseudochromosomes (plus sex chromosome and mitochondrion).All in all, this is a valuable contribution.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: I have published with Peter Holland more than three years ago, and confirm that this potential conflict of interest did not affect my ability to write an objective and unbiased review of the article.
Reviewer Expertise: Genomics, evolution, invertebrates I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
A female Nephrocerus scutellatus (specimen ID Ox001534, ToLID idNepScut1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.76, longitude -1.33) on 2021-05-31 by netting.The specimen was collected and identified by Steven Falk (University of Oxford) and snap-frozen on dry ice.The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.The idNepScut1 sample was prepared for DNA extraction

Figure 2 .
Figure 2. Genome assembly of Nephrocerus scutellatus, idNepScut1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 613,391,848 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (142,240,654 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (119,915,786 and 94,731,670 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the diptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMUPT01/dataset/CAMUPT01/snail.

Figure 3 .
Figure 3. Genome assembly of Nephrocerus scutellatus, idNepScut1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMUPT01/dataset/CAMUPT01/blob.
Protocols developed by the WSI Tree of Life core laboratory are publicly available on protocols.io(Dentonet al., 2023b).SequencingPacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from head tissue of idNepScut1 using the

Figure 4 .
Figure 4. Genome assembly of Nephrocerus scutellatus, idNepScut1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMUPT01/dataset/CAMUPT01/cumulative.

(
Uliano-Silva et al., 2023), which runs MitoFinder(Allio et al., 2020) or MITOS(Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Figure 5 .
Figure 5. Genome assembly of Nephrocerus scutellatus, idNepScut1.1:Hi-C contact map of the idNepScut1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=Ufz8JP3_TDyLNiKyEBKFdw.
submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.